The present invention relates to a novel process for preparing xcex3-decalactone.
xcex3-Decalactone, owing to its organoleptic properties, is an important aroma compound which has a fruity, peach-like flavour and aroma. In principle, xcex3-decalactone can be produced from fruits. However, it is present in these in such small amounts that it cannot be isolated economically by extraction or distillation.
Therefore, in recent years there have been numerous attempts to prepare xcex3-decalactone in biotechnological processes. Most of the processes operate with the use of various yeasts. In this case, either castor oil or the methyl ester of the ricinoleic acid isolated therefrom is converted by the yeasts. The yields achieved in this process vary between a few milligrams per litre up to 9.4 g/l in 75 hours (FR 2 734 843).
The process has, in addition, the disadvantage that a uracil-auxotrophic material is employed and thus two separate steps are necessary for the biomass formation and production.
The objection of the present invention is therefore to provide a process which makes The object of the present invention is therefore to provide a process which makes higher yields possible higher yields possible.
This object is achieved by means of the fact that a culture comprising Yarrowia lipolytica is used.
It is possible according to the invention to use Yarrowia lipolytica in a mixture with other microorganisms. However, preferably, Yarrowia lipolytica is used as a pure culture. Particular preference is given according to the invention to culturing the strain Yarrowia lipolytica HR 145 (DSM 12397).
A biologically pure culture of strain Yarrowia lipolytica, HR 145 (DSM 12397), was deposited on Aug. 26, 1998, under the terms of the Budapest Treaty at the DSMZ-Deutsche Sammiung Von Mikroorganismen Und Zellkulturen GmbH in Braunschweig under the number DSM 12397.
As substrate for the culture used according to the invention, synthetic, semisynthetic or complex culture media can be used. These comprise carbon compounds and nitrogen compounds, inorganic salts with or without trace elements and vitamins.
As carbon compounds, carbohydrates, hydrocarbons or organic base chemicals can preferably be used. Examples of compounds which can preferably be used are sugars, alcohols and/or sugar alcohols, organic acids or complex mixtures. According to the invention, preference is given to oils.
As sugar, glucose is preferably used. The useable alcohols preferably include glycerol or mannitol. Organic acids which can be used are preferably citric acid. Complex mixtures include, for example, malt extract, yeast extract, casein or casein hydrolysate. As oil, in particular castor oil is useable. In these cases, according to the invention, mixtures of two or more of the said compounds can be used.
As nitrogenous substrates, inorganic compounds can be used. Examples of these are nitrates and ammonium salts. Likewise, organic nitrogen sources can be used. These include yeast extract, soya flour, cotton seed meal, casein, casein hydrolysate, wheat gluten and corn steep liquor. It is also possible to use two or more of the said compounds as a mixture.
The inorganic salts which can be used include, for example, borates, carbonates, chlorides, molybdates, nitrates, phosphates and sulphates. As metals, the said salts preferably contain calcium, iron, potassium, cobalt, copper, magnesium, manganese, sodium or zinc. According to the invention, a mixture of two or more of the said salts can also be used.
The temperature for the culture is preferably in the range from 10 to 40xc2x0 C. Particular preference is given to the range from 20 to 35xc2x0 C., very high preference is given to 25 to 30xc2x0 C.
The pH of the medium is preferably 4 to 9. Particular preference is given to the range from 5 to 8.
During the production process, adequate aeration is necessary. The reactors which can be used according to the invention are to be designed accordingly. In principle, according to the invention, all bioreactors suitable for aerobic processes and known to those skilled in the art can thus be used. Preferably, all apparatuses suitable for any aerobic submerged process can be used. That is to say, vessels without or with a mechanical mixing device can be used according to the invention. The former include, for example, shaking apparatus, bubble-column reactors or loop reactors. The latter preferably include all known apparatuses having stirrers in any design.
The process according to the invention can be carried out continuously or batchwise. The fermentation time until a maximum amount of product is reached is in the range from 36 to 72 hours, preferably in the range from 48 to 66 hours, calculated from inoculation of the culture.
According to the invention, the substrates can be added at the beginning of the incubation, during growth or after completion of growth. This can be achieved by a single addition of substrates or by continuous successive addition during the process.
However, preference is given to continuous addition over a period of a plurality of hours after inoculation of the culture.
Using the processes described according to the invention it is surprisingly possible to produce more than 11 g/l of xcex3-decalactone in less than 70 hours. The invention and associated surprising findings are described in more detail by the following examples.